Long-chain polyunsaturated fatty acids (PUFAs), including those of the omega-3 family [also known as ω-3 (“omega-3”) fatty acids] are interesting fatty acids in nature. They are important constituents of phospholipids that play a role in decreasing membrane rigidity. Eicosapentaenoic acid (EPA) is a major constituent of the human brain's phospholipids and serves as precursor of prostaglandins and resolvins. Another important PUFA of the omega-3 family is docosahexaenoic acid (DHA). Improved cognitive and behavioural function in infant development seems correlated to high levels of this compound. For omega-3 PUFAs, and in particular for DHA and EPA, beneficial health effects have been e.g. the prevention of cancer, rheumatoid arthritis, cardiovascular diseases, the improvement of immune function, and eye and brain health [for recent overview see Teale M C (ed.) (2006) Omega-3 fatty acid research. Nova Science Publishers. New York, and references therein]. Because of these beneficial properties omega-3 PUFAs are being used extensively as nutritional lipids in health and dietary supplements and as functional ingredients in a wide range of foods. Omega-3 PUFAs presently comprise one of the biggest and strongest growing market segments in the food and beverage industry sector, with substantially increasing demand over the past years.
These days, fish oil is the most abundant and widely used natural source for omega-3 fatty acids, but named source suffers from over fishing, lack in high grade oil supply with sufficient content of DHA/EPA, and quality issues (smell, formulation challenges etc.). Alternative processes involving algae and oomycetes as producer organisms are established or under development [see overviews by Hinzpeter I et al. (2006) Grasas y Aceites 57:336-342 and Ward O P, Singh A (2005). Process Biochemistry 40:3627-3652, respectively]. Since the supply of fish oil of high quality is increasingly limited, it was attempted to find alternative, sustainable biological sources. Various groups of marine algae have been explored for over 20 years and some products based on algal biomass have meanwhile entered the market. Some oomycetes belonging to the group of stramenopiles (a group of algae-like eukaryontic organisms previously known as “Chromophyta”) were also occasionally reported to produce the above mentioned compounds (e.g. of the genera Achyla and Pythium; [Aki T et al. (1998) J Ferm Bioengin 86:504-507; Cheng M H et al. (1999) Bioresour Technol 67:101-110; Athalye S K et al. (2009) J Agric Food Chem 57:2739-2744]). In other stramenopiles (e.g. the genera Schizochytrium and Thraustochytrium; as described in U.S. Pat. No. 7,022,512 and WO2007/068997) and in species of the dinoflagellate Amphidinium (US 2006/0099694), DHA may represent up to 48% of the fatty acid content of the cells, which are the highest contents so far known in the Eukaryota. However, the cultivation of these organisms in industrial scale still poses a challenge even after several years of development.
Other alternative biological sources for omega-3 PUFAs hitherto found are prokayotic eubacteria [Nichols D et al. (1999), Curr Opin Biotechnol 10:240-246; Metz J G et al. (2001), Science 293:290-293; Gentile G et al. (2003) J Appl Microbiol 95:1124-1133]. However, the commercial exploitation of these organisms for PUFA production on an industrial scale is hampered by the slow growth characteristics of these psychrophilic microorganisms, as well as their inherently low yields and productivity. In myxobacteria, an unidentified PUFA with 20 carbon atoms and four double bonds was found first in marine genera of Plesiocystis and Enhygromyxa [Iizuka T et al. (2003) Int J Syst Evol Microbiol 53:189-195; Iizuka T. et al. (2003), Syst Appl Microbiol 26:189-196]. Recently, ARA (an omega-6 PUFA) was encountered in Phaselicystis flava, a representative of a novel myxobacterial family [Garcia R O et al. (2009), Int J Syst Evol Microbiol 50(PT12):1524-1530].
The occurrence of omega-3 PUFAs like DHA and EPA is not reported at all for Myxobacteria and so far described processes are insufficient with regard to yields, amount of PUFAs and especially concerning the production of the important omega-3 PUFAs.
Myxobacterial Taxonomy and Phylogeny
The myxobacteria are believed to have evolved as a monophyletic group of organisms in the order Myxococcales, a delta subgroup in proteobacteria. At present, 3 suborders (Cystobacterineae, Nannocystineae, and Sorangiineae) are recognized in myxobacteria [Reichenbach H (2005) Order VIII. Myxococcales Tchan, Pochon and Prevot 1948, 398AL. In Brenner D J, et al. (eds.) Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part C, pp. 1059-1072, New York: Springer]. These suborders are divided into six families, namely Cystobacteraceae, Myxococcaceae, Nannocystaceae, Kofleriaceae, Polyangiaceae, and Phaselicystidaceae.
The family Myxococcaceae is composed of the genera Myxococcus, Corallococcus and Pyxidicoccus. In the related family Cystobacteraceae, five genera are known (Cystobacter, Archangium, Hyalangium, Melittangium and Stigmatella). Nannocystaceae of the suborder Nannocsytineae are comprised of the Nannocystis and two marine genera (Enhygromyxa and Plesiocystis). Its related family Kofleriaceae is composed of the terrestrial genus Kofleria and the marine genus Heliangium. The family Polyangiaceae encompasses the genera Jahnella, Chondromyces, Polyangium, Byssovorax, and Sorangium. So far, the latter two are the only known genera of cellulose degraders among the order; most of the other taxa are difficult to isolate and cultivate. The recently discovered genus Phaselicystis is the only genus in the recently erected family Phaselicystidaceae [Garcia R O et al. (2009) Int J Syst Evol Microbiol 59:1524-1530]. At present 20 genera are recognizable and validly described in myxobacteria to cover all the known soil and marine isolates.
General Importance of 16S rDNA in Bacterial Taxonomy and Phylogeny.
16S rDNA has been widely and commonly used in bacterial systematics to designate ancestral groupings of the taxa because this gene is highly conserved between species [Weisburg W G et al. (1991) J Bacteriol 173:697-703]. In myxobacteria, the 16S rDNA phylogeny along with morphological characteristics provides a strong evidence for genetic classification [Sp{umlaut over (r)}oer C et al (1999), Int J Syst Bacteriol 49(PT 3):1255-1262]. Those myxobacterial strains assigned to the same genus by morphological classification were found to be tightly clustered in their 16S rDNA gene phylogeny. This method also provides patterns for ancestral relatedness among its member species which are reflected on the degree of phenotypic characteristics [Vellicer G J, Hillesland K (2008) In Myxobacteria: Multicellularity and Differentiation (Whitworth D E, ed.), pp. 17-40, Washington, D.C.:ASM Press].
Importance of Fatty Acid Profiles as Chemotaxonomic Markers in Bacteria
The phylogeny is in accordance with the morphological and physiological characteristics of myxobacteria. Most importantly, fatty acid profiles as inferred from GC-MS analyses of the cellular fatty acid content are generally used and deemed acceptable for taxonomic segregation of Myxobacteria, as well as many other groups of bacterial organisms, since they were found to be a constant feature, at least when standardised methodology is applied. First applications of this technique have been made in the early 1989s [Tornabenet G (1985) Methods in Microbiology 18, 209-234]. Therefore, such GC-MS (or GC-) based fatty acid profiles have been widely used in bacterial phylogeny and taxonomy. Nevertheless, a systematic approach combining both the search for particular economically important fatty acids in combination with other means of investigation to evaluate the taxonomic and phylogenetic positions of the respective fatty acid producers has so far never been carried out.
Importance of PCR-Based Methods to Explore Bacterial Species Diversity and Functional Biodiversity in Ecosystems
It has been discussed for a long time (and meanwhile proven by methods of molecular biology directed at the in situ identification of eubacteria in environmental samples) that the overall diversity of extant bacterial species is much higher than the number of known and well-described, culturable species [Amann R I et al. (1995) Microbiol Rev 59:143-169; Torsvik V et al. (1990) Appl Environ Microbiol 56:782-787]. According to current estimates, as much as 90% of the extant bacteria still remain to be discovered. Using methods such as direct sequencing of 16S rDNA from soil and other environmental samples is increasingly revealing a great diversity of DNA sequences that cannot be correlated to any of the known, culturable bacterial species. However, their phylogenetic affinities may be revealed from a homology comparison of their 16S rDNA with those of reference strains. Metagenomic techniques, which are currently under development, may in future eventually facilitate the direct utilisation of the genes and enzymes of these “unculturable” organisms. At present it remains necessary in most cases to find suitable culture conditions for the hitherto unexplored bacterial organisms and explore them at the stage of their pure cultures. As prerequisite for the characterisation of all the unexplored bacteria, as well as for their biotechnological exploitation, especial isolation techniques need to be established. This is also true in particular with regard to the discovery of novel myxobacterial taxa, as well as for various other groups of eubacteria and other microbial groups with great potential on biotechnology.
Myxobacterial groups can be specifically searched for by PCR using specific 16S rDNA primers. A previous study on a soil niche revealed at least 30 additional unknown phylogenetic groups of myxobacteria that could be detected using this approach. They are not only different from each other, but also their 16S rDNA genes differed from those of the known myxobacterial 16S rDNA gene sequences existing in GenBank and other public domain databases. These results suggested the presence of a vast undiscovered diversity of soil myxobacteria that yet remain to be cultured and explored [Zhi-Hong W. et al. (2005) Env. Microbiol. 7(10): 1602-1610].
Microbial Fermentation, with Particular Emphasis on Myxobacteria
Myxobacterial strains are usually fermented in an aqueous nutrient medium under submerged aerobic conditions. Various examples for the feasibility of large scale fermentation of this group of organisms in pilot and industrial scale are widely known to science, e.g., concerning the discovery and development of the epothilones, which have recently been approved as anticancer drugs.
After the thorough evaluation of their growth and nutrient conditions, these organisms can normally be grown well in laboratory culture and their production be scaled up in a straightforward manner. Typically, microorganisms are fermented in a nutrient medium containing a carbon source and a proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cottonseed flour, corn steep liquor, yeast, autolysed brewer's yeast with milk solids, soybean meal, cottonseed meal, corn meal, milk solids, pancreatic digest of casein, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. There is no need to add trace metals, e.g. zinc, magnesium, manganese, cobalt, iron and the like to the fermentation medium since tap water and unpurified ingredients are used as medium components.
Large scale fermentation for production cultures can be induced at any temperature conductive to satisfactory growth of the microorganisms between about 18° and 32° C. and preferably at about 28° C. Ordinarily, optimum production of compounds is obtained in about 2 to 8 days of fermentation, and preferably in about 4 to 5 days of fermentation.
Production can be carried out in shake flasks but also in solid media and stirred fermentors. When growth is carried out in shake flasks or large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form of the microorganism for inoculation. This avoids a pronounced lag in the production of the PUFA compounds and the attendant inefficient utilisation of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in an aqueous nutrient medium by inoculating this medium with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to other shake flasks or other suitable devices for fermentation of microorganisms. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilised for the production of compounds, as long as adequate growth of the microorganism is obtained.
In general, seeding of myxobacterial strains and fermentation and the production of compounds in submerged aerobic fermentation in stirred vessels is utilised. The production is independent of used containers, fermentors and starter proceedings. The compounds can also be obtained by shake-flask culture, or in other specially designed vessels such as airlift or Biowave fermentation tanks. For large volume fermentations it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating small volume of culture medium with the spore form or a lyophilised pellet of the organism. The vegetative inoculum is then transferred to a fermentation vessel where, after a suitable incubation time, compounds are produced in optimal yield.
As is customary in aerobic submerged culture process, sterile air is dispersed through the culture medium. For efficient growth of the organism, the volume of the air used is in the range of from about 0.25 to about 0.5 vvm. An optimum rate in a 10 l vessel is about 0.3 vvm with agitation provided by conventional impellers rotating at about 240 rpm. Adding of small amount (i.e. 1 ml/l) of an antifoaming agent such as silicone to fermentations media is necessary if foaming becomes a problem. For microaerophilic organisms it may be favorable to reduce the aeration further in order to support biomass production. The fermentation is usually carried out in batch mode, but to attain better growth and increased product yield, fed-batch fermentations can be carried out by supplying the required nutrient source to a growing culture once it has been depleted in the original culture medium.
The desired products will usually be present mostly in the biomass of the fermented myxobacterial strains, but in case of their overproduction, they may as well be located in the culture filtrate of the fermentation broth. The culture broth can be separated by filtering on a filter press. A variety of procedures can be employed to isolate and purify the PUFA compounds from the fermentation broth, for example, by chromatographic adsorption procedures followed by elution with a suitable solvent, column chromatography, partition chromatography, by supercritical fluid extraction, and combinations of the aforementioned methods.